lgbell at liverpool.ac.uk wrote:
>Gene S. Hall wrote:
>>>> What is the basic way to absolutely determine an unknown protein? We
>> are using SEC to separate human proteins from plasma. How can we
>> absolutely determine the molecular weight of these proteins and identify
>> them.
>> Thanks for your help,
>> Gene
>>>> --
>> %%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%
>> Prof. Gene S. Hall, Analytical Chemist Phone: 908-445-2590
>> ICPMS Laboratory FAX: 908-445-5312
>> Department of Chemistry
>> Rutgers, The State University of New Jersey
>> New Brunswick, NJ 08903
>> %%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%Mass spec, electrospray or MALDITOF of the isolated and purified protein
>would identify the mass.
>>As for the identity of the protein, mass spec of selected protease
>digests produce a 'finger print' trace. This can be compared to the mass
>spec profile of the protease digestion products of known proteins and
>hence the unknown protein identified. I beleive there are programs
>available to carry out this mapping exercise.
>>Hope this helps,
>Len Bell
>lgbell at liv.ac.uk
Depends on what you want to know.
If you have enough of the protein, a sequence is your best bet. Edman
sequencing involves coupling, cleavage, conversion to PTH amino acids then detection via mass spec or HPLC. If you can't sequence ie. if the beast is too large, you are best off doing a series of cleavages with
different proteolytic enzymes.
Bill